This invention relates to molecular biology.
One approach for elucidating protein-protein binding in cells is the yeast-based two-hybrid system (Fields and Song (1989) Nature 340:245). That system utilizes chimeric genes and detects protein-protein interactions via the activation of reporter-gene expression. Reporter-gene expression occurs as a result of reconstitution of a functional transcription factor caused by the association of fusion proteins encoded by the chimeric genes. Typically, polynucleotides encoding two-hybrid proteins are constructed and introduced into a yeast host cell. The first hybrid protein consists of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein (often referred to as the xe2x80x9cbaitxe2x80x9d). The second hybrid protein consists of the Gal4 activation domain fused to a polypeptide sequence of a second protein (often referred to as the xe2x80x9cpreyxe2x80x9d). Binding between the two-hybrid proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacZ or HIS3), which is operably linked to a Gal4 binding site.
In a first method, expression of a reporter gene that encodes a fluorescent polypeptide is used to indicate that an interaction has occurred between a bait and a prey protein. An advantage of using a fluorescent reporter polypeptide is that an interaction between a bait and prey in a mammalian cell can be readily detected, e.g., within 96 hours. In addition, the use of a fluorescent reporter polypeptide allows the identification of a single fluorescing mammalian cell without further manipulation or damage to the cell. For example, a cell that fluoresces can be identified under a fluorescent microscope. To determine the sequence of the prey protein, total DNA from a fluorescing cell is prepared, and the DNA sequence that encodes the prey amplified and sequenced.
In a second method, a prey plasmid containing an Epstein-Barr virus origin of replication (ori-P) and a bait plasmid are transfected into a mammalian cell that expresses Epstein-Barr virus nuclear antigen-1 (EBNA-1). The oriP allows the prey plasmid to replicate episomally and indefinitely in the cell. Since the prey plasmid is maintained episomally in a closed circular form, the prey plasmid can be readily introduced and recovered from a bacterial host cell.
In one aspect, the invention features a method for detecting an interaction between a bait and a prey in a mammalian cell. The method includes: (a) providing a. mammalian cell containing: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence containing a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein, including a DNA-binding domain and the bait; (iii) a prey nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and a prey; (b) incubating the cell for 96 hours or less; e.g., 72, 48, 24, or 16 hours; (c) detecting reporter gene expression, if present, thereby detecting an interaction between the bait and the prey.
The method can further include isolating total DNA from a cell expressing the reporter gene, and amplifying the nucleotide sequence that encodes the bait or prey. In one embodiment, the reporter gene is integrated into a chromosome of the cell. In another embodiment, the bait or prey is encoded by a nucleotide sequence from a nucleic acid library. The cell can be any mammalian cell, e.g., a primary, secondary or an immortalized cell such as a CV-1 cell. The reporter gene can be a fluorescent polypeptide such as a green fluorescent protein (GFP) or a blue fluorescent protein (BFP).
In another aspect, the invention features a method for detecting an interaction between a bait and a prey in a mammalian cell. The method includes: (a) providing a mammalian cell containing (i) an Epstein-Barr virus nuclear antigen-1 (EBNA-1); (ii) a reporter gene operably linked to a transcriptional regulatory sequence containing a DNA binding site for a DNA-binding domain, (iii) a bait nucleotide sequence encoding a bait fusion protein, including a DNA-binding domain and the bait (e.g., a known protein), (iv) a prey nucleotide sequence including an origin of replication for the Epstein-Barr virus nuclear antigen-1 (oriP) and encoding a prey fusion protein including a transcriptional activation moiety and the prey (e.g., an unknown protein); and (b) detecting reporter gene expression, if present, thereby detecting an interaction between the bait and the prey. The method can further include (c) isolating DNA from a cell expressing the reporter gene; and (d) recovering the nucleotide sequence including the oriP sequence, which encodes the prey fusion protein. An example of a suitable reporter gene includes a reporter gene that encodes a fluorescent protein such as a green fluorescent protein or a blue fluorescent protein. In some embodiments, the reporter gene is integrated into a chromosome of the cell. The cell can be any mammalian cell that expresses EBNA-1 (or manipulated to express EBNA-1). An example of a mammalian cell includes a cell derived from a primate or a canine. The cell can be a primary, secondary or an immortalized cell. An example of an immortalized cell is a CV-1 cell. The bait and/or the prey can be encoded by a nucleotide sequence from a nucleic acid library.
The invention also features a kit for detecting an interaction between a bait and prey in a mammalian cell. The kit includes: (a) a first gene construct which includes a regulatory sequence operably linked to a nucleotide sequence encoding a DNA-binding domain, and wherein the first gene construct includes a cloning site for inserting a nucleotide sequence encoding the bait into the first gene construct such that the bait is expressed in frame with the DNA-binding domain; (b) a second gene construct which includes: an oriP sequence, a regulatory sequence operably linked to a nucleotide sequence encoding a transcriptional activation domain, and wherein the second gene construct includes a cloning site for inserting a nucleotide sequence encoding the prey into the second gene construct such that the prey is expressed in frame with the transcriptional activation domain; (c) a mammalian cell that expresses an EBNA-1, including a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for the DNA-binding domain, wherein the reporter gene expresses the fluorescent polypeptide when the bait and prey interact; and (d) instructions for use.
Also within the scope of the invention is a method of identifying an agent that disrupts interaction between a bait and a prey, including: (a) providing a mammalian cell having: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for a DNA-binding domain, (ii) a first nucleotide sequence encoding a bait fusion protein, including a DNA-binding domain and the bait, (iii) a second nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and the prey; (b) contacting the mammalian cell with a test agent; (c) incubating the cell for 96 hours or less, e.g., 72, 48, 24 or 16 hours; and (d) detecting a decrease in expression of the reporter gene compared to the level of expression of the reporter gene in a mammalian control cell, if present, thereby detecting an agent that disrupts interaction between the bait and the prey.
The invention further features a method of identifying an agent that enhances interaction between a bait and a prey, including: (a) providing a mammalian cell having: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein including a DNA-binding domain and the bait, (iii) a prey nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and a prey; (b) incubating the cell for a period of time, e.g., 96 hours or less e.g., 78, 48, 24, or 16 hours; and (c) detecting an increase in expression of the reporter gene compared to the level of expression of the reporter gene in a mammalian control cell, thereby detecting an agent that enhances interaction between the bait and the prey.
The invention further features a method of identifying an agent that enhances interaction between a bait and a prey, including: (a) providing a mammalian cell having: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein including a DNA-binding domain and the bait, (iii) a prey nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and a prey; (b) incubating the cell for a period of time, e.g., 96 hours or less e.g., 78, 48, 24, or 16 hours; and (c) detecting an increase in expression of the reporter gene compared to the level of expression of the reporter gene in a mammalian control cell, thereby detecting an agent that disrupts interaction between the bait and the prey.
As used herein, xe2x80x9cDNA-binding domainxe2x80x9d means an amino acid sequence that binds specifically to a particular DNA sequence. The site where the DNA-binding domain binds is known as a DNA binding site.
As used herein, xe2x80x9ctranscriptional activation domainxe2x80x9d means an amino acid sequence which when in proximity to transcriptional regulatory DNA elements of a target gene, activates gene transcription.
As used herein, a xe2x80x9creporter genexe2x80x9d means a gene whose expression can be assayed.
As used herein, xe2x80x9cinteractorxe2x80x9d means a protein which is able to form a complex with another protein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents and other references mentioned herein are incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed-description and the claims.